Combined HS6ST1 and HS6ST2 loss disrupts the formation and migration of gonadotropin-releasing hormone neurons

6-O sulfotransferase (HS6ST) enzymes modify heparan sulfate proteoglycans (HSPGs) to promote growth factor binding to extracellular matrix or receptors. HS6ST1 variants are reported in patients with GnRH deficiency (GD), which is caused by the defective development of gonadotropin releasing hormone (GnRH) neurons. However, it is unknown whether and how HS6ST family members cooperate in GnRH neuron development. Here, we show that Hs6st1 and Hs6st2 are co-expressed in the nasal neurogenic epithelia, where GnRH neurons and their migratory substrate, the terminal nerve, originate. The combined but not individual disruption of Hs6st1 and Hs6st2 disrupted vomeronasal organ formation with incomplete penetrance and impaired brain entry of GnRH neurons, similar to mice with loss of the morphogen FGF8 or the axon guidance cue SEMA3A, respectively. In agreement with a role for HS6ST-modified HSPG in regulating SEMA3A signalling, SEMA3A binding to nasal tissues was impaired in the absence of HS6ST1 and HS6ST2. Our results establish a mechanistic role for HS6ST1 in GD and suggest HS6ST2 as an additional susceptibility locus for GD.